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Characterisation of Glycosylation Patterns Utilizing a DNA-Sequenzer and Mass Spectrometry

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons86442

Rapp,  E.
Physical and Chemical Foundations of Process Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86475

Schwarzer,  J.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86448

Reichl,  U.
Otto-von-Guericke-Universität Magdeburg;
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Rapp, E., Schwarzer, J., & Reichl, U. (2007). Characterisation of Glycosylation Patterns Utilizing a DNA-Sequenzer and Mass Spectrometry. Talk presented at 14th European Carbohydrate Symposium. Lübeck, Germany. 2007-09-02 - 2007-09-07.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-9773-E
Abstract
The presented approach, allows the characterization of N-glycosylation patterns of glycoproteins. This is shown exemplarily by viral proteins of influenza viruses, replicated in mammalian cell-culture for the purpose of vaccine-production. The envelope of influenza contains two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The functional role of their glycans is still not completely understood, but it is known, that structural modifications of these glycans can influence viral replication dynamics and immune response after vaccination. The glycosylation pattern of viral proteins can be affected by the virus strain, by the glycosylation machinery of the host cell, by cultivation conditions in upstream processing and via incipient degradation of the glycoproteins during virus inactivation and downstream processing. Hence, the ability of monitoring and thereby controlling the glycosylation pattern during the virus production process, is a prerequisite for yield enhancement in vaccine production and to ensure sufficient immune-response after vaccination. Within this work, the N-glycans are analyzed in two stages: first comparing glycan-pool fingerprints and second - structural identification via database matching and additional sequenzing of the glycans. For the generation of fingerprints we are utilizing a DNA-sequenzer based on capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). The developed procedure allows monitoring of N-glycosylation patterns of relevant glycoproteins during the major steps of up- and downstream processing in influenza virus vaccine production, achieving a limit of detection down to the zeptomolar range. Besides generation of glycan-pool fingerprints, additional structural information can be obtained, assigning peaks of these fingerprints to normalized data of our homebuilt database (i.e. a series of N-glycans with known structures, already measured with via CGE-LIF and cross-checked via mass spectrometry). Further structural analysis of the HA N-glycans is done by consecutive sequencing the glycan ladder utilizing reagent array analysis method (RAAM) in combination with CGE-LIF and mass spectrometry.